PROJECT SUMMARY Though deaths from heart disease and stroke have fallen dramatically in the last 40 years, these thrombotic diseases are still our largest killer. The overall goal of this grant is to develop a new class of anti-platelet compounds for managing the risk and treatment of thrombosis. Platelets play a critical role in thrombosis, the major cause of mortality and morbidity. Medical management of acute coronary syndrome and cerebrovascular injury is centered on anti-platelet therapies. Thrombin is an essential activator of protease activated receptors (PAR) in platelets for generation of a hemostatic plug. Human platelets have both PAR1, the high-affinity thrombin receptor that underlies hemostasis at the vessel wall, and PAR4, the low-affinity thrombin receptor which is activated slowly in the clot because it requires substantial thrombin build up. Our guiding hypothesis is that PAR4 is an attractive target for anti-platelet therapy in thrombosis and cerebrovascular injury, because of its local activation at the site of the 3D clot as more thrombin builds up. We hypothesize that PAR4 antagonism might minimize clot build-up in the vessel, but not affect hemostasis as potently and thus may decrease bleeding side effects and be a better therapeutic anti-platelet target. Blacks have a significantly higher incidence of heart disease and stroke and worse survival. Recently, a hyperactive PAR4 allele was discovered and found to occur in 63% of blacks but only 16% of whites. We have used HTS and medicinal chemistry to obtain nM and pM affinity antagonists of PAR4 which block both alleles. We propose to optimize bioavailability, potency, and selectivity of our lead molecules for both of these PAR4 alleles and test them in a baboon shunt model of thrombosis. We will 1) optimize the best series of PAR4 antagonists using a library approach, 2) determine selectivity and potency of compounds, and carry forward compounds that have diverse modes of action to maximize safety 3) determine the pharmacokinetics, metabolism and disposition of these PAR4 antagonists in vitro in baboon and human and in vivo in baboon, and 4) test the optimized compound in human platelets under flow and in a baboon shunt model of thrombosis. Completion of these aims will provide evidence in primate models of thrombosis that PAR4 antagonism is safe and efficacious. The long-term goal is to demonstrate PAR4 as an alternative target for preventive and therapeutic suppression of pathologic thrombus formation. An antagonist that is active against the hyperactive PAR4 allele may have the potential to develop into an ideal anti-platelet agent that can decrease the racial disparity of outcomes of thrombotic events.